Cell cycle assay

DLD-1, HT29 and HCT116 cells were seeded to six-well plates at a density of 3×10⁵, 4×10⁵ and 3.5×10⁵ cells per well, respectively. Cells were starved in serum-free medium (RPMI-1640 or DMEM) for 48 h. A total of 24 h before the end of starvation, miR-NC or miR-1291 was transfected at a final concentration of 30 nM. Cells were collected at the indicated times (0, 12, 24 and 48 h) and fixed in 70% ethanol for 30 min at 4°C. After fixation, cells were washed twice with PBS and incubated with RNase (Sigma Aldrich; Merck KGaA) for 20 min at 37°C. Cells were treated with propidium iodide (PI; Dojindo Molecular Technologies, Inc.) for 20 min on ice and analyzed by flow cytometry (Spectral Analyzer SA3800; Sony Biotechnology, Inc.) with SA3800 2.0 software (Sony Biotechnology, Inc.).